Fabrication off immunosensor for detection of poultry virus (nghiên cứu chế tạo cảm biến miễn dịch điện hóa để phát hiện virut cúm gia cầm)

MINISTRY OF EDUCATION AND TRAINING HANOI UNOVERSITY OF TECHNOLOGY AND SCIENCE INTERNATIONAL TRAINING INSTITUTE FOR MATERIALS SCIENCE - TRAN QUANG THINH FABRICATION OF IMMUNOSENSOR FOR DETECTION OF POULTRY VIRUS MASTER THESIS OF MATERIALS SCIENCE Batch ITIMS-2014 SUPERVISOR Assoc Prof Mai Anh Tuan Dr Nguyen Hien Hanoi – 2016 CONTENTS LIST OF ABBREVIATIONS LIST OF TABLES LIST OF FIGURES Chapter IMMUNOSENSOR AND IMMUNE REACTION .8 1.1 Biosensor and immunosensor 1.1.1 Electrochemical immunosensor 1.1.1.1 Transducer 10 1.1.1.2 Bioreceptor 12 1.1.2 Indirect and direct immunosensor 12 1.2 Immune Reaction 14 1.2.1 Structure of antibody 14 1.2.2 The principle of antibody-antigen interaction 17 1.2.3 Monoclonal and polyclonal antibody .23 1.2.4 Immunoglobulin IgG and IgY 23 Chapter FABRICATION OF IMMUNOSENSOR 26 2.1 Antibody Immobilization Approaches 26 2.1.1 Physical adsorption 27 2.1.2 Covalent attachment 28 2.1.3 Bio-affinity .32 2.2 Fabrication of electrochemical sensor based on gold thin film electrodes 35 2.2.1 Photomask design .35 2.2.2 Main processes in the electrochemical sensor fabrication .36 2.2.3 Sensor pretreatment 40 2.3 Antibody Immobilization 41 2.3.1 Antibody Immobilization using PrA/GA approach 42 2.3.2 Antibody Immobilization using SAM/NHS approach .43 2.4 Immunoassay Protocol 46 Chapter DETECTION OF NEWCASTLE DISEASE VIRUS USING ELECTROCHEMICAL IMMUNOSENSOR 47 3.1 Characteristics of electrochemical sensor .47 3.2 Characteristics of PrA-GA immunosensor .50 3.2.1 Cyclic voltammetry characterization of PrA-GA immunosensor 50 3.2.2 Effect of the IgY concentration on the immobilization of PrA-GA immunosensor 53 3.3 Characteristics of SAM-NHS immunosensor .54 3.3.1 Cyclic voltammetry characterization of SAM-NHS immunosensor .55 3.3.2 Effect of the pH value on the immobilization of SAM-NHS immunosensor 58 3.4 Stability of the signal of ND virus immunosensors 59 3.5 Detection of Newcastle disease virus .61 3.4.1 Effect of the immunoreaction time 62 3.5.2 Sensitivity of Newcastle disease virus immunosensor .63 CONCLUSION 68 REFERENCE 69 PUBLICATION .74 LIST OF ABBREVIATIONS BSA Bovine serum albumin CDR Complementarity-determining region CE Counter electrode CV Cyclic Voltammetry DCC N,N'-Dicyclohexylcarbodiimide DNA Deoxyribonucleic acid EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EID50 50 Percent Embryo Infectious Dose EIS Electrochemical Impedance Spectroscopy GA Glutaraldehyde IgG Immunoglobulin G IgY Immunoglobulin Y LOD Limit of detection LOQ Limit of quantification ND Newcastle Disease NDV Newcastle Disease virus NHS N-hydroxysuccinimide PCR Polymerase chain reaction PrA Protein A RE Reference electrode TGA Thioglycolic acid SAM Self-assembled monolayer SD Standard deviation WE Working electrode LIST OF TABLES Table 1.1 Properties of immunoglobulin classes Table 2.1 Sputtering parameters Table 3.1 The crucial parameters obtained from experimental CV data for fabrication procedures of immunosensor Table 3.2 Experimental conditions for the attachment of components Table 3.3 The crucial parameters obtained from experimental CV data for fabrication procedures of immunosensor Table 3.4 Experimental conditions for the attachment of components Table 3.5 The average and standard deviation of Ipeak of sensors Table 3.6 The crucial parameters obtained from the calibration Table 3.7 Comparison of analytical properties of different immunosensors for the detection of Avian Influenza LIST OF FIGURES Figure 1.1 The performing principle of electrochemical immunosensor Figure 1.2 Direct and Indirect immunosensor Figure 1.3 (A) Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab [18], (B) The schematic description of the structure of an IgG antibody, (C) The domain structure of an IgG antibody Figure 1.4 X-ray crystallography of the interactions between Fab of 1C1 antibody and EphA2 antigen Figure 1.5 Non-covalent bonds in the antigen-antibody interaction Figure 1.6 The structural difference between IgG and IgY Figure 2.1 Different orientations of the antibody immobilized on the substrate Figure 2.3 Pre-treated substrate with maleimide and antibody immobilization by thiol groups Figure 2.4 Covalent attachment through carbohydrate residues of antibody Figure 2.5 Biotinylation of antibody by NHS reagent Figure 2.6 Avidin-biotin affinity for immobilization Figure 2.7 Protein A/G-mediated bio-affinity immobilization Figure 2.8 ssDNA-antibody conjugation to form a hydrazone linker Figure 2.9 Structure of the integrated electrode Figure 2.10 Photomask design and detailed structure of electrode sensor Figure 2.11 Main processes for sensor fabrication Figure 2.12 Image of electrochemical sensors on a wafer and a complete sensor Figure 2.13 Electrochemical cleaning and activation of electrodes in sulfuric acid by CV Figure 2.14 The schematic description of the fabrication procedures of PrA-GA immunosensor Figure 2.15 The schematic of antibody immobilization process using SAM-NHS Figure 3.1 CV curves of sensor with commercial Ag/AgCl RE and Ag/AgCl wire Figure 3.2 The uniform of sensors Figure 3.3 The reaction of GA linker with protein A and IgY antibody Figure 3.4 CV characterization of modified electrode recorded on Au electrode Figure 3.5 Effect of the antibody concentration Figure 3.6 The main reactions on the antibody immobilization Figure 3.7 CV characterization of modification of WE Figure 3.8 The schematic description of the CV responses of modified electrode Figure 3.9 Effect of pH value of the immobilization of antibody Figure 3.10 The average and the SD of Ipeak of the bare Au electrode Figure 3.11 The schematic description of the ND virus detection mechanism Figure 3.12 Effect of the immunoreaction time Figure 3.13 (A) The CV curves of PrA-GA immunosensor (a) in buffer solution and after assay with (b) 102, (c) 103, (d) 104, (e) 105, (f) 106 EID50/mL ND virus (B) the relationship between ΔIpeak and various ND virus concentrations of PrA-GA immunosensor Figure 3.14 The relationship between ΔIpeak and various ND virus concentrations INTRODUCTION Newcastle disease (ND) is one of the most popular infection diseases in poultry that widely spreads in Southern East Asian countries, including Vietnam Its most notable effect is that causes severe economic losses in domestic poultry due to its highly contagion, especially in chicken Over the past years, the conventional qualitative methods (haemagglutination inhibition, agar gel precipitation test and Latex agglutination test) as well as semi-quantitative analysis (enzyme-linked immunosorbent assay and immunofluorescence test) were introduced for clinical diagnosis of ND Although these methods allow effective determinations ND virus in infective samples, which require rather complicated procedures for sample preparations and sophisticated instruments for assays Thus, it is necessary to develop methods that offer a simple, rapid, cost-effective analytical strategy, which can be easily used for applications in contamination studies of ND To investigate infection diseases, the fabrication and application of electrochemical immunosensor have been considerably developed However, most of the works have used monoclonal immunoglobulin G (antibody IgG) from mammalian blood Egg yolk immunoglobulin (IgY) from chickens can be employed as an alternate IgG in immunoassay, which offers some advantages with respect to animal care, high productivity and special suitability in the source of antibodies In our work, electrochemical immunosensor using IgY as receptors in configuration has been developed to detect ND virus This thesis is organized into three chapters: In the first chapter, the basic concepts about immunosensor and fundamental theory of immune reaction will be introduced In the second chapter, the fabrication of electrochemical immunosensor will be described in detail In the last chapter, the characterization of immunosensor carried out with ND virus will be discussed Chapter IMMUNOSENSOR AND IMMUNE REACTION 1.1 Biosensor and immunosensor A biosensor is an analyte device consisting of a biological sensing element attached a signal transducer, which converts signals of the biological reactions into measurable signals [1] The biological sensing element ranges from oligonucleotides (DNA or RAN) to enzymes, proteins, cells, antibodies or antigens Transducer designed on a solid-state substrate that plays a role converting the signals recorded from biological sensing element into measurable signals like the electric signals Biological reactions are able to lead to that include the changing of pH value, electronic or ionic transfer, refraction, luminescence, micro mass or thermal transfer… The biosensors based on antibodies or antigens are known as immunosensors Thus, the four most common kind of immunosensors based on the signal of biological reactions are optical, electrochemical, micro mass and thermal [2] North [3] proposed the first concept of the immunosensor in 1985 in which the bioelement was antibody Recently, the term immnosensors were described as the ones that can convert the specific antibody-antigen interactions into measurable signals In principle, either antibodies or an antibody-antigen complexes immobilized on transducer’s surface play the role as a bio-receptor toward a target element (another antibody or antigen) Most of the immunosensors are designed that based on the two mechanisms such as biological catalysis and biological affinity The biological catalysts are usually enzymes catalyzing for biochemical reactions, while the biological affinity bases on the specific interaction of proteins, lectins, receptors, live cells, nucleic acids, antibodies and antigens [2] The applications of the biosensor and immunosensor comprise a wide range of tasks, ranging from clinical diagnostics, food safety, industrial processes control, pollution monitoring, drug discovery, to military and security applications [4] The interest in the fields of biosensors is reflected directly in its fast rise in the number of publications In 1985, there were approximately 100 papers on this subject and this number rose to 4500 in 2011 Furthermore, the papers published in 2011 alone represented more than 10% of all articles ever published concerning the biosensors This upward trend can also be seen in the global market for biosensors which increased from billion US dollars market share in 2000 to 13 billion dollars and predictions for 2018 show figures around 17 billion dollar mark [5] 1.1.1 Electrochemical immunosensor According to the IUPAC suggestion of definition for electrochemical biosensors [6], an immunosensor is an integrated device consisting of an immunochemical recognition element in direct spatial contact with a transducer element Electrochemical immunosensors employ either antibodies or their complementary binding partners, i.e antigens or haptens as biological recognition elements in combination with electrodes or field-effect transistors Advantage of this kind of immunosensor ranges from low sample consumption, reasonable cost of instrumentations to miniaturization possibility, which are the main reasons for extensive development of electrochemical immunosensors Figure 1.1 The performing principle of electrochemical immunosensor Ipeak(0) values In the addition, the Ipeak(0) should be high enough, that is better for the detection of virus at low concentrations 𝛥𝐼𝑝𝑒𝑎𝑘 = 𝐼𝑝𝑒𝑎𝑘 (0) − 𝐼𝑝𝑒𝑎𝑘 (𝑖) 𝐼𝑝𝑒𝑎𝑘 (0) (3.1) where Ipeak(0) is the Ipeak of immunosensor, and the Ipeak(i) is its Ipeak obtained after the immunoreaction with ND virus Herein, both kinds of immunosensor are investigated stability of the signal through the average and standard deviation (SD) (n = 5) of Ipeak(0) A comparison of the Ipeak(0) between two kinds of immunosensor is shown in Fig 3.10 The Ipeak of bare Au electrode is also shown to observe the changes of signal after the modifications Figure 3.10 The average and the SD of Ipeak of the bare Au electrode (A), PrA-GA immunosensor (B), and SAM-NHS immunosensor (C) From the Fig 3.10 and the table 3.5, both kinds of immunosensor exhibit high uniformity due to their quite small SD values In comparison, the Ipeak(0) of SAM-NHS immunosensor is considerably higher than that of PrA-GA immunosensor 60 Table 3.5 The average and standard deviation of Ipeak of sensors (n=5) The average (μA) SD Bare Au electrode 197.1 ± 7.70 (3.9%) PrA-GA immunosensor 88.2 ± 1.03 (1.1%) SAM-NHS immunosensor 120.3 ± 3.1 (2.5%) Sensor Thus, both kinds of immunosensor are good in stability of the signal In the prediction, SAM-NHS immunosensor may be better for the virus detection due to its higher Ipeak(0) 3.5 Detection of Newcastle disease virus As mentioned in the above sections, when anti-ND virus antibody and supporting substances are attached on the sensor’s WE, the senor becomes an electrochemical immunosensor In this study, the biological target to be detected by the electrochemical immunosensor is the inactivated ND virus The virus detection is based on a direct way of which Fe(CN)63-/4- is used as an electroactive marker without adding any other labeling substance Figure 3.11 The schematic description of the ND virus detection mechanism 61 The detection of ND virus is carried out by CV measurements before and after immunosensor treated with ND virus The ΔIpeak calculated by the formula 3.3, which determines the change of peak currents, is used to evaluate the performance of immunosensor The ND virus detection mechanism of the label-free electrochemical immunosensor can be described as Fig 3.11 As the description in Fig 3.11, when ND virus is specifically attached on the antibody layer, the electro-transfer of Fe(CN)63-/4- is hindered by the combination This leads to a decrease of the peak current on the CV curve 3.4.1 Effect of the immunoreaction time Immunoreaction time, which is determined by the incubation time of ND virus, is a very important parameter effecting the performance of immunosensor when detection of virus In this section, we will investigate to give the effective experimental immunoreaction time for each kind of fabricated immunosensor PrA-GA immunosensor PrA-GA immunosensors were incubated with ND virus samples at the same 106 EID50/ml concentration and the tested immunoreaction time varied from to 90 From the results in Fig 3.12B, when the virus incubation time is 60 min, the ΔIpeak reached a maximum, indicating a maximum immunoreactive quantity of ND viruses on the immunosensor This is referred to the complete specific binding of ND virus with anti-ND virus antibody 62 Figure 3.12 Effect of the immunoreaction time SAM-NHS immunosensor SAM-NHS immunosensors were incubated with ND virus samples at the same 102 EID50/ml concentration and the tested incubation time varied from 15 to 90 As the results shown in Fig 3.12B, a maximum ΔIpeak value is also observed when the incubation time at 60 When the incubation time is longer than 60 min, slight decreases of the ΔIpeak is observed from both kinds of immunosensor The reason may be that the immunoreaction is reversible, and reaches equilibrium when a largest amount of formed immune complexes at 60 Because our assay is carried out in an open system, outside factors will significantly effect to the equilibrium After 60 min, the equilibrium moves slowly towards the reverse reaction that is the dissociation of immune complex, and reduces the amount of viruses binding with antibody of immunosensor This leads to a increase of Ipeak(i) as well as a decrease of ΔIpeak Therefore, 60 is chosen as an effective incubation time of ND virus samples, which will be used for both kinds of immunosensor 3.5.2 Sensitivity of Newcastle disease virus immunosensor Herein, quantitative assessment of sensitivity of two kinds of immunosensor is performed using inactivated ND virus solution in PBS buffer from 1×100 to 1×106 EID50/mL at room temperature for h To observe a correlation between Ipeak and ΔIpeak, CV curves as well as ΔIpeak values, which measured in immunoassay between PrA-GA immunosensor and various ND virus concentrations, are shown in Fig 3.12 From Fig 3.12A, peak currents decrease gradually with the increasing concentration of ND virus, indicating the inhibition effects of ND virus on the electron-transfer between Fe(CN)63-/4- and PrA-GA immunosensor Correspondingly, in Fig 3.12B, the 63 increase of ΔIpeak is exhibited by a nearly linear relationship between ΔIpeak and the logarithm of the concentration of ND virus Figure 3.13 (A) The CV curves of PrA-GA immunosensor (a) in buffer solution and after assay with (b) 102, (c) 103, (d) 104, (e) 105, (f) 106 EID50/mL ND virus (B) the relationship between ΔIpeak and various ND virus concentrations of PrA-GA immunosensor To determine the sensitivity of both kinds of immunosensor, the calibration curves, that expressing the relationship between ΔIpeak and the logarithm of ND virus concentration at each kind of immunosensor, are given in Fig 3.14 The curves, determined in the data treatment by Origin software, show the average values of five measurements with error bars showing the standard deviation of five measurements at each data points The crucial parameters obtained from the calibration are also exhibited in Table 3.6 Table 3.6 The crucial parameters obtained from the calibration Parameter PrA-GA SAM-NHS Linear range of concentration 102-106 EID50/ml 102-106 EID50/ml Correlation coefficient (R2) 0.983 0.991 Slope (S) 0.0289 0.0588 Standard deviation of the slope (SDS) 0.00188 0.00286 y-intercept (yi) -0.0099 0.2606 64 Standard deviation of y-intercept (σ) 0.0080 0.0119 LOD 0.95 (9 EID50/ml) 0.67 (5 EID50/ml) LOQ 2.88 (103 EID50/ml) 2.03 (102 EID50/ml) From Fig.3.14, both kinds of immunosensor exhibit the nearly linear relationship between ΔIpeak and lgCNDvirus in the range of 102 EID50/ml – 106 EID50/ml The linear regression equation of PrA-GA immunosensor is ΔIpeak = 0.0289lgCNDvirus – 0.0099 (CNDvirus denoting concentration at EID50/ml, R2 = 0.983), and that of SAM-NHS immunosensor is ΔIpeak = 0.0588lgCNDvirus + 0.261(R2 =0.991) Figure 3.14 The relationship between ΔIpeak and various ND virus concentrations In the comparison, the SAM-NHS immunosensor has the higher slope value of the calibration curve, as well as the higher ΔIpeak values at each concentration point, than those of PrA-GA immunosensor According to Michael Swartz’s report [5], the limits of detection (LOD) and the limits of quantification (LOQ) can be calculated using the equations: 65 𝐿𝑂𝐷 = 3.3 × 𝜎 𝑆 (3.4) 𝐿𝑂𝑄 = 10 × 𝜎 𝑆 (3.5) where σ is the standard deviation of the response y-intercepts of the regression lines and S is the slope of the calibration curve In our work, in case of PrA-GA immunosensor the LOD and LOQ values obtained are 100.95 EID50/ml and 103 EID50/ml, respectively In case of SAM-NHS immunosensor LOD and LOQ are calculated to be 100.67 EID50/ml and 102 EID50/ml, respectively Thus, the results, containing sensitivity, LOD, and LOQ, indicate that the sensitivity of SAM-NHS immunosensor is higher than that of PrAGA immunosensor Table 3.7 Comparison of analytical properties of different immunosensors for the detection of Avian Influenza Technique Immobilization Type Antibody Virus Detection limit Ref Optic APTES/CDI Indirect Goat monoclonal IgY Purified NDV ng/ml [53] RT-PCR Inactivated NDV 5×102 ELD50/ml [59] RT-PCR Inactivated NDV 5×102 ELD50/ml [52] RT-PCR Inactivated NDV 105.8 ELD50/ml [58] RRT-PCR NDV RNA 101 EID50/ml [60] IgY from egg yolk Inactivated NDV 100.95 EID50/ml IgY from egg yolk Inactivated NDV 100.67 (EID50/ml) CV CV PrA/GA SAM/DCC/NHS Direct Direct CV: cyclic voltammetry, NDV: Newcastle disease virus, CDI: carbonyldiimidazole, RBCs: red blood cells, AI: Avian influenza, EID: Embryo Infectious Dose, ELD: Embryo Lethal Dose, FTA card: Flinders Technology Associates filter paper, RT-PCR reverse transcriptase-polymerase chain reaction, RRT-PCR: real-time reverse-transcription PCR In the same conditions of measurement as well as of immune reaction (temperature, pH, time), a high sensitivity of immunosensor is closely related to a 66 This work high density of specifically active antibody on sensor’s WE Thus, from the higher sensitivity observed at SAM-NHS immunosensor, we can conclude that the antibody immobilization using SAM and NHS is better for the performance of ND virus immunosensor Table 3.7 summarizes the published articles based on different techniques and immunoassay procedures, which were used to detect ND virus As can be seen, PCR (polymerase chain reaction) with striking sensitivity is commonly employed in immune biology for detection of ND virus as well as quantitative determination of ND virus vaccine In the relatively comparison, the detection limit of PrA-GA immunosensor and SAM-NHS immunosensor are, respectively, 100.95 and 100.67 EID50/ml, indicating that they are the good sensitivity immunosensors In addition to the sensitivity, obviously, the main advantages of the electrochemical immunosensors proposed here are their simple fabrication, with the possibility for miniaturization, small sample volume, direct detection, and the utilization of IgY antibody from egg yolk This study also reveals that egg yolk immunoglobulin (IgY) from chickens can be employed as an alternate IgG in immunoassays as well as immunosensor, which offers some advantages with respect to animal care, high productivity and special suitability in the source of antibodies 67 CONCLUSION Sensor integrating three gold electrodes was designed and fabricated by using simple planar process on silicon wafer Electrochemical characterization of sensor was investigated by cyclic voltammetry (CV) measurement, which shows that sensor has stable performance, high uniformity, and easy operation The fabricated sensor has been used as platform for development of two kinds of immunosensor in detecting Newcastle disease virus (ND virus) One immunosensor is based on successive attachment of protein A, glutaradehyde, and then anti-ND virus IgY The other one is composed by the direct formation of covalent bonds between SAM and anti-ND virus IgY through coupling agent (NHS ester) Effect of experiment conditions for each kind of immunosensor was also mentioned The results show that both approaches can be used effectively for immobilization of anti-ND virus IgY Based on the electrochemical response when immune reaction occurs, immunosensors were employed to detection various concentration samples of ND virus vaccine The result shows that both kinds of immunosensor have the linear detection range of 102 EID50/ml – 106 EID50/ml In comparison, SAM-NHS immunosensor exhibits the higher sensitivity because of its higher slope value of the calibration curve as well as lower LOD value In the future, we will widen detectable target range of these immunosensors by using real ND virus samples extracted from 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TaqMan Minor Groove Binder Probes”, J Clin Microbiol, 47, pp 2114–2123 61 T H Tang, C E Lunte, H H Brian (1988), “P.aminophenyl phosphate: an improved substrate for electrochemical enzyme immnoassay”, Anal Chim Acta., 214(1), pp 187–195 62 T.V.V Quân (2016), “Development of electrochemical micro.system towards the application in Biomedical Analysis”, Master of Science, Hanoi University of Science and Technology 63 Z Jie, D Liping, D Ling (2014), “An ultrasensitive electrochemical immunosensor for carcinoembryonic antigen detection based on staphylococcal protein A—Au nanoparticle modified gold electrode”, Sensors Actuators B Chem., 197, pp 220–227 73 PUBLICATION Tran Quang Thinh, Tran Thi Luyen, Chu Thi Xuan, Tran Quang Huy, Ho Phung Ha, Mai Anh Tuan, An Immobilized Method of The IgY Antibody Based The Immunosensor Detecting The Newcastle Disease Virus, The 3rd International Conference on Advance Materials and Nanotachnology (ICAMN) 2016, pp 314318 74 ... processes of bio-components onto the sensor’s WE to form immunosensors For detection of Newcastle disease virus (ND virus) in poultry, anti-Newcastle disease virus immunoglobulin Y (anti-ND virus. .. characterization of SAM-NHS immunosensor .55 3.3.2 Effect of the pH value on the immobilization of SAM-NHS immunosensor 58 3.4 Stability of the signal of ND virus immunosensors 59 3.5 Detection. .. areas of chemistry From obtained 24 characteristic of CV measurements, we will develop the electrochemical immunosensor towards quantitative detection of ND virus 25 Chapter FABRICATION OF IMMUNOSENSOR